This is a proposal to investigate mechanisms of cranial patterning and the pathophysiology of of craniosynostosis, a human developmental defect characterized by the premature fusion of calvarial bones. Recent findings in human genetics have demonstrated that mutations in several genes can produce craniosynostosis syndromes. We showed that an activating mutation in the homeodomain protein Msx2 causes craniosynostosis, Boston type. Several groups have demonstrated that activating mutations in FGF receptors 1-3 cause Crouzon, per, Jackson-Weiss, and Pfeiffer syndromes. Loss of function mutations in the basic HLH protein M-twist are responsible for Saethre-Chotzen syndrome. Despite the identification of specific genetic defects that cause craniosynostosis, the cellular an developmental mechanisms underlying this disorder re poorly understood. In this proposal, we focus on these unresolved issues through an investigation of the function of the Msx2 and twist genes. In this proposal, we focus on these unresolved issues through an analysis of the function of the Msx2 and twist genes. We based our proposal on several key findings. First, gain of function and loss of function phenotypes in the mouse suggest a critical and complex role for the Msx2 and twist genes in calvarial morphogenesis, and in the differentiation of calvarial osteogenic cells. Second, a variety of studies suggest that Msx and twist genes are likely to function in growth factor mediated signaling in calvarial development-Msx genes in the BMP and possibly FGF pathways, twist in the FGF pathway. Third, our preliminary data with the Kedes group suggest that the Kedes group suggest that the Msx2 and twist proteins can interact physically and functionally (Project 2). These data are the foundation of our overall hypothesis that Msx2 and regulate calvarial morphogenesis through effects on specific cell populations in the calvarial plates and sutures, that these effects re mediated by BMP and FGF signaling, and that a synergistic interaction between Msx2 and twist is a key aspect of this regulation. These are our specific aims: First, we will document in greater detail now normal cranial development is altered in Msx and twist mutant mice, and we will use chimera analysis to identify the tissues in which Msx2 and twist are required for calvarial development Third, we will develop structure-function assays for Msx2 and twist in calvarial development. Third, we will develop structure-function assays for Msx2 and twist in calvarial development, and ultimately test the hypothesis that a synergistic interaction between Msx2 and twist is required for calvarial development. This work will provide fundamental information about the molecular genetics calvarial development and may explain how mutations in three different classes of genes, Msx, fgfr, and twist-produce craniosynostosis in humans.